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Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability,with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia,coarsened facial features and intellectual disability,due to truncating mutations in the sorting nexin gene SNX14,encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate,a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma,accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction. View Publication

BACKGROUND Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years,the molecular mechanism accounting for this activity is still unclear. OBJECTIVES AND METHODS To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions,using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. RESULTS We identified 328 annotated genes differentially regulated by anagrelide,including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3,whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4,a known TRIB3 inducer. Notably,immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α,which is an upstream regulator of ATF4,and increased ATF4 protein levels. Furthermore,salubrinal,an inhibitor of eIF2α dephosphorylation,increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. CONCLUSIONS These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis. View Publication

The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously,we identified a human pluripotent stem-cell-specific lectin probe,called rBC2LCN,by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A,termed rBC2LCN-PE23,which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells,followed by its internalization,allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus,rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies. View Publication

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However,NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study,we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation,apoptosis,gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay,flow cytometry,terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining,Western Blot and a xenograft mouse model,respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells,stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR,p-EGFR,p-STAT3,Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover,the combined treatment of TG101348 and erlotinib induced apoptosis,inhibited the activation of p-EGFR and p-STAT3,and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment. View Publication

An investigation was conducted to demonstrate that neurodazine (Nz) and neurodazole (Nzl),two imidazole-based small molecules,promote neuronal differentiation in both neuroblastoma and fibroblast cells. The results show that differentiated cells generated by treatment with Nz and Nzl express neuron-specific markers. The ability of Nz and Nzl to induce neurogenesis of neuroblastoma and fibroblast cells was found to be comparable to those of the known neurogenic factors,retinoic acid and trichostatin A. In addition,the cells differentiated by Nz and Nzl are observed to express different isoforms of glutamate receptors. The results of signaling pathway studies reveal that two substances enhance neurogenesis in neuroblastoma cells by activating Wnt and Shh signaling pathways and neurogenesis in fibroblast cells by mainly activating the Wnt signaling pathway. Observations made in the present study suggest that Nz and Nzl will serve as chemical tools to generate specific populations of neuronal cells from readily available and simply manageable cells. View Publication

Insulin-like growth factor 1 (IGF-1) enhances cellular proliferation and reduces apoptosis during the early differentiation of bone marrow derived mesenchymal stem cells (BMSCs) into neural progenitor-like cells (NPCs) in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). BMSCs were differentiated in three groups of growth factors: (A) EGF + bFGF,(B) EGF + bFGF + IGF-1,and (C) without growth factor. To unravel the molecular mechanisms of the NPCs derivation,microarray analysis using GeneChip miRNA arrays was performed. The profiles were compared among the groups. Annotated microRNA fingerprints (GSE60060) delineated 46 microRNAs temporally up-regulated or down-regulated compared to group C. The expressions of selected microRNAs were validated by real-time PCR. Among the 46 microRNAs,30 were consistently expressed for minimum of two consecutive time intervals. In Group B,only miR-496 was up-regulated and 12 microRNAs,including the let-7 family,miR-1224,miR-125a-3p,miR-214,miR-22,miR-320,miR-708,and miR-93,were down-regulated. Bioinformatics analysis reveals that some of these microRNAs (miR-22,miR-214,miR-125a-3p,miR-320 and let-7 family) are associated with reduction of apoptosis. Here,we summarize the roles of key microRNAs associated with IGF-1 in the differentiation of BMSCs into NPCs. These findings may provide clues to further our understanding of the mechanisms and roles of microRNAs as key regulators of BMSC-derived NPC maintenance. View Publication

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Non-viral gene delivery into human embryonic stem cells (hESCs)is an important tool for controlling cell fate. However,the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632),a small molecule that inhibits the rho-associated protein kinase pathway,is utilized to induce transient colony spreading which leads to increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection,cells can revert back to its normal state and do not show alteration of pluripotency. This approach provides a simple,effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation. View Publication

Chronic obstructive pulmonary disease (COPD) is a highly prevalent,chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-$$ activity can modify inflammatory responses in several models of lung injury,the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema,with excessive macrophage accumulation associated with increased expression of chemokines,Ccl5,Cxcl10,and Cxcl15. Conversely,treatment of mice with a pharmacological PPAR$$ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro,CS increased lung epithelial cell chemokine expression in a PPAR$$ activation-dependent fashion. The ability of PPAR$$ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPAR$$-mediated transrepression of NF-$$B activity. Pharmacological or genetic activation of PPAR$$ activity abrogated CS-dependent induction of NF-$$B activity. Regulation of NF-$$B activity involved direct PPAR$$-NF-$$B interaction and PPAR$$-mediated effects on IKK activation,I$$B$$ degradation,and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-$$B-dependent,CS-induced chemokine-mediated regulation of inflammatory cell accumulation. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases,both to study them and to explore therapies. However,a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study,we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV),adenoviruses and lentiviral vectors in hESC,hiPSC,and the derived cardiomyocytes. In undifferentiated cells,adenoviral and lentiviral vectors were superior,whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes,1,2,6,and 9,of which 2 and 6 were superior in their transduction efficiency. Interestingly,we observed that AAVs severely diminished the viability of undifferentiated cells,an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore,we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally,adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion,adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines,whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells. View Publication

Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications,technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype,three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified,cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research. View Publication

The biological effects of low-dose ionizing radiation (LDIR) exposure in humans are not comprehensively understood,generating a high degree of controversy in published literature. The earliest stages of human development are known to be among the most sensitive to stress exposures,especially genotoxic stresses. However,the risks stemming from exposure to LDIR,particularly within the clinical diagnostic relevant dose range,have not been directly evaluated in human embryonic stem cells (hESCs). Here,we describe the dynamics of the whole genome transcriptional responses of different hESC lines to both LDIR and,as a reference,high-dose IR (HDIR). We found that even doses as low as 0.05 Gy could trigger statistically significant transient changes in a rather limited subset of genes in all hESCs lines examined. Gene expression signatures of hESCs exposed to IR appear to be highly dose-,time-,and cell line-dependent. We identified 50 genes constituting consensus gene expression signature as an early response to HDIR across all lines of hESC examined. We observed substantial differences in biological pathways affected by either LDIR or HDIR in hESCs,suggesting that the molecular mechanisms underpinning the responses of hESC may fundamentally differ depending on radiation doses. View Publication